1 3D Bioprinting Bioinks: an up-to-date overview 1 3D bioprinting has emerged as a transformative technology in the fields of regenerative medicine and tissue engineering, empowering the precise fabrication of complex biological structures. At the core of this innovation is the creation of bioinks that merge living cells with biomaterials to produce scaffolds that replicate the natural extracellular matrix. This review explores the various types of bioinks, including natural, synthetic, and hybrid formulations, also their unique properties such as rheological behavior, biocompatibility, degradability, and printability. Recent advancements in bioink development, particularly the rise of smart bioinks that react to environmental stimuli and bioinks customized for specific tissues, are discussed in detail. Additionally, the applications of 3D bioprinting with bioinks are examined, including their roles in tissue engineering, drug delivery systems, organ printing, and regenerative medicine. This study aims to guide researchers and practitioners in selecting optimal bioinks for specific bioprinting applications, ultimately contributing to the translation of 3D bioprinted tissues into clinical practice. 1 14 Aramis Moradi Zand Institute of Higher Education, Shiraz, Fars Iran aramismoradi66@gmail.com Negin Khosravi School of science and health, The University of Georgia, Tbilisi Georgia Negin.kvi76@gmail.com Bhekumuzi Sfundo Khanyile Mathematics and Physics, Faculty of Applied Sciences, Cape Peninsula University of Technology, Bellville Campus; Symphony Way, Bellville,South, Cape Town, 7530 South Africa sfkhalilr@kfu.edu.sa Fariborz Sharifianjazi Center for Advanced Materials and Structures, School of Science and Technology, The University of Georgia, P.O. Box. 0171, Tbilisi Georgia f.sharifianjazi@ug.edu.ge Matin Sorkhabi School of science and health, The University of Georgia, Tbilisi Georgia Matinsorkhabi@ug.edu.ge 3D Bioprinting Bioinks Biocompatibility Biomaterials Biocompatibility Drug Delivery Systems [1] T.D. Ngo, A. Kashani, G. Imbalzano, K.T.Q. Nguyen, D. Hui, Additive manufacturing (3D printing): A review of materials, methods, applications and challenges, Composites Part B: Engineering 143 (2018) 172-196.##[2] S. Vanaei, M.S. Parizi, S. Vanaei, F. Salemizadehparizi, H.R. Vanaei, An Overview on Materials and Techniques in 3D Bioprinting Toward Biomedical Application, Engineered Regeneration 2 (2021) 1-18. ##[3] Z. Gu, J. Fu, H. Lin, Y. He, Development of 3D bioprinting: From printing methods to biomedical applications, Asian Journal of Pharmaceutical Sciences 15(5) (2020) 529-557.##[4] Z. Xie, M. Gao, A.O. Lobo, T.J. Webster, 3D Bioprinting in Tissue Engineering for Medical Applications: The Classic and the Hybrid, Polymers 12(8) (2020) 1717. ##[5] V.B. Veeravalli R S, Savani S, et al. (July 10, 2023) Three-Dimensional Bioprinting in Medicine: A Comprehensive Overview of Current Progress and Challenges Faced. Cureus 15(7): e41624.##[6] K. Pushparaj, B. Balasubramanian, M. Pappuswamy, V. Anand Arumugam, K. Durairaj, W.-C. Liu, A. Meyyazhagan, S. Park, Out of Box Thinking to Tangible Science: A Benchmark History of 3D Bio-Printing in Regenerative Medicine and Tissues Engineering, Life 13(4) (2023) 954.##[7] P.G. Manita, I. Garcia-Orue, E. Santos-Vizcaino, R.M. Hernandez, M. Igartua, 3D Bioprinting of Functional Skin Substitutes: From Current Achievements to Future Goals, Pharmaceuticals 14(4) (2021) 362.##[8] S. Ji, M. Guvendiren, Complex 3D bioprinting methods, APL Bioengineering 5(1) (2021) .##[9] P.S. Gungor-Ozkerim, I. Inci, Y.S. Zhang, A. Khademhosseini, M.R. Dokmeci, Bioinks for 3D bioprinting: an overview, Biomaterials Science 6(5) (2018) 915-946 .##[10] M. Mobaraki, M. Ghaffari, A. Yazdanpanah, Y. Luo, D.K. Mills, Bioinks and bioprinting: A focused review, Bioprinting 18 (2020) e00080 .##[11] C. Benwood, J. Chrenek, R.L. Kirsch, N.Z. Masri, H. Richards, K. Teetzen, S.M. Willerth, Natural Biomaterials and Their Use as Bioinks for Printing Tissues, Bioengineering (Basel) 8(2) (2021).##[12] G. Rijal, Bioinks of Natural Biomaterials for Printing Tissues, Bioengineering (Basel) 10(6) (2023).##[13] S. Ghosh, H.-G. Yi, A Review on Bioinks and their Application in Plant Bioprinting, IJB 8(4) (2022) .##[14] A. Veiga, I.V. Silva, M.M. Duarte, A.L. Oliveira, Current Trends on Protein Driven Bioinks for 3D Printing, Pharmaceutics 13(9) (2021) 1444.##[15] P. Kozlov, G. Burdygina, The structure and properties of solid gelatin and the principles of their modification, Polymer 24(6) (1983) 651-666.##[16] M. Wang, W. Li, J. Hao, A. Gonzales, Z. Zhao, R.S. Flores, X. Kuang, X. Mu, T. Ching, G. Tang, Z. Luo, C.E. Garciamendez-Mijares, J.K. Sahoo, M.F. Wells, G. Niu, P. Agrawal, A. Quiñones-Hinojosa, K. Eggan, Y.S. Zhang, Molecularly cleavable bioinks facilitate high-performance digital light processing-based bioprinting of functional volumetric soft tissues, Nature Communications 13(1) (2022) 3317 .##[17] A. Fatimi, O.V. Okoro, D. Podstawczyk, J. Siminska-Stanny, A. Shavandi, Natural Hydrogel-Based Bio-Inks for 3D Bioprinting in Tissue Engineering: A Review, Gels 8(3) (2022) 179.##[18] P. Dorishetty, N.K. Dutta, N.R. Choudhury, Bioprintable tough hydrogels for tissue engineering applications, Advances in Colloid and Interface Science 281 (2020) 102163.##[19] Z. Gan, X. Qin, H. Liu, J. Liu, J. Qin, Recent advances in defined hydrogels in organoid research, Bioactive Materials 28 (2023) 386-401.##[20] X. Pang, X. Zhuang, Z. Tang, X. Chen, Polylactic acid (PLA): research, development and industrialization, Biotechnology journal 5(11) (2010) 1125-1136.##[21] Y. Ramot, M. Haim-Zada, A.J. Domb, A. Nyska, Biocompatibility and safety of PLA and its copolymers, Advanced drug delivery reviews 107 (2016) 153-162.##[22] K. Willson, A. Atala, J.J. Yoo, Bioprinting au natural: the biologics of bioinks, Biomolecules 11(11) (2021) 1593.##[23] D.K. Chandra, R.L. Reis, S.C. Kundu, A. Kumar, C. Mahapatra, Nanomaterials-Based Hybrid Bioink Platforms in Advancing 3D Bioprinting Technologies for Regenerative Medicine, ACS Biomaterials Science & Engineering (2024).##[24] Y. Chen, Y. Chen, X. Xiong, R. Cui, G. Zhang, C. Wang, D. Xiao, S. Qu, J. Weng, Hybridizing gellan/alginate and thixotropic magnesium phosphate-based hydrogel scaffolds for enhanced osteochondral repair, Materials Today Bio 14 (2022) 100261.##[25] Y. Chen, X. Xiong, X. Liu, R. Cui, C. Wang, G. Zhao, W. Zhi, M. Lu, K. Duan, J. Weng, 3D Bioprinting of shear-thinning hybrid bioinks with excellent bioactivity derived from gellan/alginate and thixotropic magnesium phosphate-based gels, Journal of Materials Chemistry B 8(25) (2020) 5500-5514.##[26] Y. Chen, X. Xiong, X. Liu, R. Cui, C. Wang, G. Zhao, W. Zhi, M. Lu, K. Duan, J. Weng, S. Qu, J. Ge, 3D Bioprinting of shear-thinning hybrid bioinks with excellent bioactivity derived from gellan/alginate and thixotropic magnesium phosphate-based gels, Journal of Materials Chemistry B 8(25) (2020) 5500-5514.##[27] J. Yang, Z. Li, S. Li, Q. Zhang, X. Zhou, C. He, Tunable metacrylated silk fibroin-based hybrid bioinks for the bioprinting of tissue engineering scaffolds, Biomaterials Science 11(5) (2023) 1895-1909.##[28] M.T. Sultan, O.J. Lee, J.S. Lee, C.H. Park, Three-dimensional digital light-processing bioprinting using silk fibroin-based bio-ink: recent advancements in biomedical applications, Biomedicines 10(12) (2022) 3224.##[29] D. Kim, M. Kim, J. Lee, J. Jang, Review on Multicomponent Hydrogel Bioinks Based on Natural Biomaterials for Bioprinting 3D Liver Tissues, Frontiers in Bioengineering and Biotechnology 10 (2022).##[30] N. Kasula, P. Dhokare, A. Bhattacharyya, I. Noh, Recent Advances in 3D Bioprinting of Polysaccharide-based Bioinks for Fabrication of Bioengineered Tissues, Molecular Systems Design & Engineering (2024).##[31] K. Hölzl, S. Lin, L. Tytgat, S. Van Vlierberghe, L. Gu, A. Ovsianikov, Bioink properties before, during and after 3D bioprinting, Biofabrication 8(3) (2016) 032002.##[32] I.I. Gungor-Ozkerim PS , Zhang YS , Khademhosseini A , Dokmeci MR . Bioinks for 3D bioprinting: an overview. Biomater Sci. 2018;6(5):915-946.##[33] J.H.Y. Chung, S. Naficy, Z. Yue, R. Kapsa, A. Quigley, S.E. Moulton, G.G. Wallace, Bio-ink properties and printability for extrusion printing living cells, Biomaterials Science 1(7) (2013) 763-773 .##[34] P. Ezhilarasi, P. Karthik, N. Chhanwal, C. Anandharamakrishnan, Nanoencapsulation techniques for food bioactive components: a review, Food and bioprocess technology 6 (2013) 628-647.##[35] A. Ahmadi Soufivand, J. Faber, J. Hinrichsen, S. Budday, Multilayer 3D bioprinting and complex mechanical properties of alginate-gelatin mesostructures, Scientific Reports 13(1) (2023) 11253.##[36] Y. Yang, X. Song, X. Li, Z. Chen, C. Zhou, Q. Zhou, Y. Chen, Recent progress in biomimetic additive manufacturing technology: from materials to functional structures, Advanced Materials 30(36) (2018) 1706539.##[37] A. Schwab, R. Levato, M. D’Este, S. Piluso, D. Eglin, J. Malda, Printability and shape fidelity of bioinks in 3D bioprinting, Chemical reviews 120(19) (2020) 11028-11055.##[38] R.F. Pereira, A. Sousa, C.C. Barrias, P.J. Bártolo, P.L. Granja, A single-component hydrogel bioink for bioprinting of bioengineered 3D constructs for dermal tissue engineering, Materials Horizons 5(6) (2018) 1100-1111.##[39] Z. Liu, M.A. Meyers, Z. Zhang, R.O. Ritchie, Functional gradients and heterogeneities in biological materials: Design principles, functions, and bioinspired applications, Progress in Materials Science 88 (2017) 467-498.##[40] M. Tavafoghi, M.A. Darabi, M. Mahmoodi, R. Tutar, C. Xu, A. Mirjafari, F. Billi, W. Swieszkowski, F. Nasrollahi, S. Ahadian, Multimaterial bioprinting and combination of processing techniques towards the fabrication of biomimetic tissues and organs, Biofabrication 13(4) (2021) 042002.##[41] N. Chopra, Multifunctional and multicomponent heterostructured one-dimensional nanostructures: advances in growth, characterisation, and applications, Materials Technology 25(3-4) (2010) 212-230.##[42] L. Mascia, M. Xanthos, An overview of additives and modifiers for polymer blends: facts, deductions, and uncertainties, Advances in Polymer Technology: Journal of the Polymer Processing Institute 11(4) (1992) 237-248.##[43] T. Mandal, R. Sylla, H.U. Bahia, S. Barmand, Effect of cross-linking agents on the rheological properties of polymer-modified bitumen, Road Materials and Pavement Design 16(sup1) (2015) 349-361.##[44] C. Yu, J. Jiang, A Perspective on Using Machine Learning in 3D Bioprinting, Int J Bioprint 6(1) (2020) 253.##[45] S. Derakhshanfar, R. Mbeleck, K. Xu, X. Zhang, W. Zhong, M. Xing, 3D bioprinting for biomedical devices and tissue engineering: A review of recent trends and advances, Bioactive materials 3(2) (2018) 144-156.##[46] S. Vijayavenkataraman, W.-C. Yan, W.F. Lu, C.-H. Wang, J.Y.H. Fuh, 3D bioprinting of tissues and organs for regenerative medicine, Advanced drug delivery reviews 132 (2018) 296-332.##[47] P. Ramiah, L.C. du Toit, Y.E. Choonara, P.P.D. Kondiah, V. Pillay, Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration, Frontiers in Materials 7 (2020).##[48] B.S. Kim, Y.W. Kwon, J.-S. Kong, G.T. Park, G. Gao, W. Han, M.-B. Kim, H. Lee, J.H. Kim, D.-W. Cho, 3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: A step towards advanced skin tissue engineering, Biomaterials 168 (2018) 38-53 .##[49] T. Zidarič, M. Milojević, L. Gradišnik, K. Stana Kleinschek, U. Maver, T. Maver, Polysaccharide-Based Bioink Formulation for 3D Bioprinting of an In Vitro Model of the Human Dermis, Nanomaterials, 2020.##[50] L.A. Sharpe, A.M. Daily, S.D. Horava, N.A. Peppas, Therapeutic applications of hydrogels in oral drug delivery, Expert opinion on drug delivery 11(6) (2014) 901-915.##[51] J. Yin, M. Yan, Y. Wang, J. Fu, H. Suo, 3D Bioprinting of Low-Concentration Cell-Laden Gelatin Methacrylate (GelMA) Bioinks with a Two-Step Cross-linking Strategy, ACS Applied Materials & Interfaces 10(8) (2018) 6849-6857.##[52] D. Williams, P. Thayer, H. Martinez, E. Gatenholm, A. Khademhosseini, A perspective on the physical, mechanical and biological specifications of bioinks and the development of functional tissues in 3D bioprinting, Bioprinting 9 (2018) 19-36.##[53] A. Yaneva, D. Shopova, D. Bakova, A. Mihaylova, P. Kasnakova, M. Hristozova, M. Semerdjieva, The progress in bioprinting and its potential impact on health-related quality of life, Bioengineering 10(8) (2023) 910.##[54] R.R. Jose, M.J. Rodriguez, T.A. Dixon, F. Omenetto, D.L. Kaplan, Evolution of bioinks and additive manufacturing technologies for 3D bioprinting, ACS biomaterials science & engineering 2(10) (2016) 1662-1678.##[55] H. Jo, S. Brito, B.M. Kwak, S. Park, M.-G. Lee, B.-H. Bin, Applications of Mesenchymal Stem Cells in Skin Regeneration and Rejuvenation, International Journal of Molecular Sciences, 2021.##[56] H. Budharaju, H. Chandrababu, A. Zennifer, D. Chellappan, S. Sethuraman, D. Sundaramurthi, Tuning thermoresponsive properties of carboxymethyl cellulose (CMC)–agarose composite bioinks to fabricate complex 3D constructs for regenerative medicine, International Journal of Biological Macromolecules 260 (2024) 129443.##[57] I. Donderwinkel, J.C. Van Hest, N.R. Cameron, Bio-inks for 3D bioprinting: recent advances and future prospects, Polymer Chemistry 8(31) (2017) 4451-4471.##[58] Q. Gao, J.-S. Lee, B.S. Kim, G. Gao, Three-dimensional printing of smart constructs using stimuli-responsive biomaterials: A future direction of precision medicine, International Journal of Bioprinting 9(1) (2022) 638.##[59] R. Suntornnond, J. An, C.K. Chua, Bioprinting of thermoresponsive hydrogels for next generation tissue engineering: a review, Macromolecular Materials and Engineering 302(1) (2017) 1600266.##[60] P. Heidarian, A.Z. Kouzani, A. Kaynak, M. Paulino, B. Nasri-Nasrabadi, Dynamic hydrogels and polymers as inks for three-dimensional printing, ACS Biomaterials Science & Engineering 5(6) (2019) 2688-2707.##[61] I. Choi, S. Jang, S. Jung, S. Woo, J. Kim, C. Bak, Y. Lee, S. Park, A dual stimuli-responsive smart soft carrier using multi-material 4D printing, Materials Horizons 10(9) (2023) 3668-3679 .##[62] N. Faramarzi, I.K. Yazdi, M. Nabavinia, A. Gemma, A. Fanelli, A. Caizzone, L.M. Ptaszek, I. Sinha, A. Khademhosseini, J.N. Ruskin, Patient‐specific bioinks for 3D bioprinting of tissue engineering scaffolds, Advanced healthcare materials 7(11) (2018) 1701347.##[63] D.G. O'Shea, C.M. Curtin, F.J. O'Brien, Articulation inspired by nature: a review of biomimetic and biologically active 3D printed scaffolds for cartilage tissue engineering, Biomaterials Science 10(10) (2022) 2462-2483.##[64] B.S. Kim, S. Das, J. Jang, D.-W. Cho, Decellularized extracellular matrix-based bioinks for engineering tissue-and organ-specific microenvironments, Chemical reviews 120(19) (2020) 10608-10661.##[65] S. Karuppusamy, G. Rajauria, S. Fitzpatrick, H. Lyons, H. McMahon, J. Curtin, B.K. Tiwari, C. O’Donnell, Biological Properties and Health-Promoting Functions of Laminarin: A Comprehensive Review of Preclinical and Clinical Studies, Marine Drugs, 2022.##[66] Z. Wang, W. Kapadia, C. Li, F. Lin, R.F. Pereira, P.L. Granja, B. Sarmento, W. Cui, Tissue-specific engineering: 3D bioprinting in regenerative medicine, Journal of Controlled Release 329 (2021) 237-256.##[67]Z. Maan, N.Z. Masri, S.M. Willerth, Smart Bioinks for the Printing of Human Tissue Models, Biomolecules, 2022.##[68] F. Tasnim, A. Sadraei, B. Datta, M. Khan, K.Y. Choi, A. Sahasrabudhe, T.A. Vega Gálvez, I. Wicaksono, O. Rosello, C. Nunez-Lopez, Towards personalized medicine: the evolution of imperceptible health-care technologies, foresight 20(6) (2018) 589-601.#[69] Y. Fang, Y. Guo, M. Ji, B. Li, Y. Guo, J. Zhu, T. Zhang, Z. Xiong, 3D Printing of Cell-Laden Microgel-Based Biphasic Bioink with Heterogeneous Microenvironment for Biomedical Applications, Advanced Functional Materials 32(13) (2022) 2109810. [70] N. Diamantides, C. Dugopolski, E. Blahut, S. Kennedy, L.J. Bonassar, High density cell seeding affects the rheology and printability of collagen bioinks, Biofabrication 11(4) (2019) 045016.##[71] R.F. Pereira, B.N. Lourenço, P.J. Bártolo, P.L. Granja, Bioprinting a Multifunctional Bioink to Engineer Clickable 3D Cellular Niches with Tunable Matrix Microenvironmental Cues, Adv Healthc Mater 10(2) (2021) e2001176.##[72] A. Pössl, D. Hartzke, T.M. Schmidts, F.E. Runkel, P. Schlupp, A targeted rheological bioink development guideline and its systematic correlation with printing behavior, Biofabrication 13(3) (2021) 035021.##[73] M. Ali, A.K. Pr, J.J. Yoo, F. Zahran, A. Atala, S.J. Lee, A Photo-Crosslinkable Kidney ECM-Derived Bioink Accelerates Renal Tissue Formation, Advanced Healthcare Materials 8(7) (2019) 1800992.##[74] M. Nowicki, W. Zhu, K. Sarkar, R. Rao, L.G. Zhang, 3D printing multiphasic osteochondral tissue constructs with nano to micro features via PCL based bioink, Bioprinting 17 (2020) e00066.##[75] D.K. Chandra, R.L. Reis, S.C. Kundu, A. Kumar, C. Mahapatra, Nanomaterials-Based Hybrid Bioink Platforms in Advancing 3D Bioprinting Technologies for Regenerative Medicine, ACS Biomaterials Science & Engineering 10(7) (2024) 4145-4174.##[76] A. Nadernezhad, O.S. Caliskan, F. Topuz, F. Afghah, B. Erman, B. Koc, Nanocomposite Bioinks Based on Agarose and 2D Nanosilicates with Tunable Flow Properties and Bioactivity for 3D Bioprinting, ACS Applied Bio Materials 2(2) (2019) 796-806.